Single-molecule imaging of aquaporin-4 array dynamics in astrocytes

Aquaporin-4 (AQP4) facilitates water transport across astrocytic membranes in the brain, forming highly structured nanometric arrays. AQP4 has a central role in regulating cerebrospinal fluid (CSF) circulation and facilitating the clearance of solutes from the extracellular space of the brain. Adrenergic signaling has been shown to modulate the volume of the extracellular space of the brain via AQP4 localized at the end-feet of astrocytes, but the mechanisms by which AQP4 regulates CSF inflow and outflow in the brain remain elusive. Using advanced imaging techniques, including super-resolution microscopy and single-molecule tracking, we investigated the hypothesis that β-adrenergic receptor activation induces cellular changes that regulate AQP4 array size and mobility, thus influencing water transport in the brain. We report that the β-adrenergic agonist, isoproterenol hydrochloride, decreases AQP4 array size and enhances its membrane mobility, while hyperosmotic conditions induce the formation of larger, less mobile arrays. These findings reveal that AQP4 arrays are dynamic structures, responsive to adrenergic signals and osmotic changes, highlighting a novel regulatory mechanism of water transport in the brain. Our results provide insights into the molecular control of CSF circulation and extracellular brain space volume, laying the groundwork for understanding the relationship between astrocyte water transport, sleep physiology, and neurodegeneration.


AAV purificaCon:
Following a 120-hour long incubaDon, the AAV-producing cells were harvested and AAV parDcles were isolated using Takara Maxi prep kit (Takara, #6678), following manufacturer's instrucDon.Briefly, the cells were detached through a 10 min incubaDon with 6.25 mM EDTA (pH 7.5, FisherScienDfic #10135423), pelleted by centrifugaDon at 2000x g for 10 min.The cell pellet was resuspended in AAV extracDon soluDon A plus and incubated for 5 min at RT.Following a 10 min 5000g centrifugaDon at 4°C, the supernatant was treated with AAV ExtracDon soluDon B. Cryonase Cold-acDve Nuclease was added to the extract and incubated for 1 h at 37°C, ater which Precipitator A was applied and incubated for 30min at 37°C.Ater the addiDon of Precipitator B, the mixture was vortexed and centrifuged at 5000x g for 5 min at 4°C.The supernatant was filtered through a Millex-HV 0.45 μm filter unit and transferred to an Amicon Ultra-4, 100kDa filter.The filter with the sample was centrifuged at 2000x g for 5 minutes at 15°C six Dmes, adding fresh suspension buffer each Dme.The viral parDcles in the Amicon filter were resuspended and stored as single-use aliquots at -80°C.The Dver of each viral prep was determined using AAV real-Dme PCR DtraDon kit (Takara, #6233).

Transduction
Mixed corDcal cultures were transduced at day in vitro 1 (DIV=1) by supplemen1ng with fresh growth medium containing AAV parDcles (at 1 -2.3*10^10 genome copies/mL).Half of the medium was replaced ater 72 h and subsequent half medium changes were done every second day, unDl cultures reached DIV=9 and experimental treatments took place.
We used isoproterenol hydrochloride (Sigma-Aldrich #I6504), a β-adrenergic agonist, to modulate adrenergic signalling in primary corDcal cultures.Isoproterenol hydrochloride (ISO) was diluted to a 1 mM stock soluDon in sterile deionised water and kept at -80°C.Prior to experimental treatment, the agonist was diluted to a 1 µM final concentraDon in culture medium.

AQP4 dSTORM and cluster analysis
For dSTORM experiments primary cultures (DIV = 9) grown on 18 mm coverslips were treated with 1 µM ISO or vehicle control for 1 h, immunostained as described above, and stored in PBS at 4°C in the dark unDl imaging.
The chamber was sealed off with a coverslip (VWR #631-0153P) to eliminate oxygen and thereby enhance fluorophore stability.Imaging was performed using a commercial TIRF microscope (Oxford Nanoimaging Ltd) fived with an Olympus 1.4 NA 100x oil immersion super apochromaDc objecDve and the laser illuminaDon angle was set to 52 allowing for TIRF.Glial fibrillary acidic protein (GFAP) was iniDally imaged with standard GFP se€ngs before proceeding with STORM imaging of AQP4.SequenDal imaging and acDvaDon of Alexa Fluor 647 tagging AQP4 was done with a 630 nm laser (314 W/cm 2 ) and a 405 nm laser (212 W/cm 2 ), respecDvely.Streams of 200 images of Alexa Fluor 647 with an exposure Dme of 33 ms per frame were intercalated by acDvaDon pulses of 6.6 s with the 405 nm laser.These steps were repeated 40 Dmes per field of view.
ResoluDon and localizaDon number calculaDons were performed using the RustFRC python package as described previously 2 and the script is available at hvps://doi.org/10.5281/zenodo.7290477.
Custer analysis was performed using CODI, an Oxford Nanoimager developed sotware.To correct for potenDal sample drit during the acquisiDon, an inbuilt drit correcDon 3 was performed.Each localizaDon was fived to a 2D Gaussian distribuDon and any of those with a photon count lower than 5000 and or localizaDon precision larger than 15 nm were discarded.Cluster analysis was done with density based spaDal clustering of applicaDons with noise (DBSCAN).Each cluster needed to have at least 15 localizaDons and each localizaDon had to be within 150 nm of each other.The cluster area in nm 2 was ploved as a cumulaDve frequency, which allows to compare different sized samples and binning was set to 100 nm 2 .

Quantum dot tracking in mixed cortical cultures
To determine the mobility of endogenous AQP4, the receptor was labelled with Quantum Dots (QD) similar to a previously described procedure 4  Streams of QD images were processed in Fiji 5 with the plugin TrackMate 6 over regions of interest (ROIs) imaged on GFP-posiDve astrocytes.A Laplacian of Gaussian (Log) filter and a median filter were used and sub-pixel localizaDon were enabled.The detected maxima in the filtered image (first frame) were saved and used to calculate the number of QDs per µm 2 .A simple linear assignment problem (LAP) tracker with a linking max distance of five pixels, gap-closing max distance of five pixels and gap-closing max frame gap of four was used.Trajectories were subsequently analysed in Matlab R2019b (Mathworks) to obtain the mean-squared displacement and instantaneous diffusion coefficients for each trajectory as previously described. 7The minimal length of analysed tracks was set to 50 frames, and the diffusion coefficient of each track was calculated from linear fits of the first five points of the plot of mean squared displacement as a funcDon of lag Dme over the trajectory.All diffusion coefficients were ploved as cumulaDve frequency distribuDons, with the binning set to 0.001 µm 2 /s.Arrays were considered immobile when the diffusion coefficient was smaller than 0.001 µm 2 /s.Fluorescence intensity over Dme of QD were obtained by tracking QD with the Spot DetecDon Tool of IMARIS 9.9.

Statistical analyses
StaDsDcal analysis was performed on Prism 10.9.3 (GraphPad).To compare two individual groups, an unpaired, two-tailed t-test was used.However, if data did not pass the normality test (Shapiro-Wilk), we opted for Mann-Whitney test.CumulaDve distribuDons were compared using Kolmogorov-Smirnov.A pvalue below 0.05 was considered to be staDsDcally significant.